- Email: [email protected]
841-2 Periadventitial fat inflammation correlates with plaque inflammation in patients with coronary plaque ruptures: New marker of plaque vulnerability?
JACC
March 3, 2004
ABSTRACTS - Vascular Disease, Hypertension, and Prevention 505A
R2
increased to 0.44 (P<0.05). Finally, a model which included WHR, Head fat, and Upper Body fat yielded a maximal R2 of 0.47 (P<0.05), an improvement over either set of measurements alone. Conclusion: Simple measures when used alone are as effective as more sophisticated methods like DEXA for predicting SI in a healthy population. However, when these measures are combined there is a small increased predictive power.
Parameter
Estimate
t Ratio
Pr > t
R2
Weight (kg)
-0.020
-11.47
<0.0001
0.37
BMI
-0.062
-12.77
<0.0001
0.40
Head % Fat
-0.040
-12.52
<0.0001
0.41
Upper Body Fat (kg)
-0.044
-12.50
<0.0001
0.41
Head Fat (kg)
-0.807
-12.78
<0.0001
0.42
Waist (cm)
-0.028
-13.38
<0.0001
0.42
* Age and gender were forced into every model.
1124-196
Impact of Elevated Age and Sex-Adjusted Body Mass Index in School Age Children on Insulin Resistance and Lipoprotein Subfractions
David K. Murdock, Karen Olson, Kirk Jenkins, Mary Roth, Sharon Fox, Patrick Hughes, Bryan Hendricks, Cardiovascular Research and Education Foundation, Inc, Wausau, WI, Community Health Care/Wausau Health Foundation, Wausau, WI Background: The Wausau SCHOOL Project is a community-based effort to assess the frequency of cardiovascular risk factors in students in the Wausau School District. Objective: Define the incidence of elevated BMI and determine its relationship to standard lipid profiles, homeostatic model assessment of insulin resistance (HOMA-IR), LDL particle size and number. Methods: Age and sex adjusted BMI (zBMI), fasting plasma insulin, glucose levels and nuclear magnetic resonance lipid profiles (LipoScience®) was measured in 225 randomly selected students (110 in 2nd grade and 125 in 11th grade). Overweight was defined as zBMI > 85th centile based on 2000 CDC norms. Results: Over 31% of the students were classified as overweight. (30.6% in 2nd grade and 32.5% in 11th grade). HOMA-IR values were significantly higher for 11th graders than for 2nd graders (2.18±2.65 vs. 1.22± 1.07, p<0.01). HOMA-IR values were also significantly higher for females than for males (2.04±2.66 vs. 1.33±0.94, p<0.05). Those with zBMI above the 85th centile had significantly higher levels of total cholesterol (182.0±34.0 vs. 170.2±28.5, p<0.01), LDL (123.4±28.5 vs. 112.7±25.7, p<0.01), triglycerides (118.6±80.7 vs. 86.4±35.6, p<0.001), LDL particle number (1299.8±379.5 vs. 1135±248.0, p<0.001), and HOMA-IR (2.61±3.47 vs. 1.32±0.09, p< 0.001), while HDL levels were lower (43.7±10.0 vs. 47.5 ±9.7, p<0.01). There were no significant differences in LDL particle size. Conclusions: A significant percentage of children and adolescents are overweight. IR increases significantly with age and zBMI and is associated with an atherogenic lipid profile characterized by an increase in the number of small LDL particles and TG levels and lower HDL levels, similar to that seen in adults.
1124-197
Additive Gene-Gene Interaction Between CYP7A1 and Apolipoprotein E as Genetic Determinants of LowDensity Lipoprotein Cholesterol-Lowering Response to Atorvastatin
Kouji Kajinami, Noboru Takekoshi, Margaret E. Brousseau, Jose M. Ordovas, Ernst J. Schaefer, Kanazawa Medical University, Uchinada, Japan, Tufts University, Boston, MA Background: The mechanisms responsible for interindividual variation in response to statin therapy remain uncertain. Bile acid biosynthesis is one of the determinants of intracellular cholesterol and, in turn, cholesterol synthesis rate in hepatocytes. This raises the hypothesis that variation in the cholesterol 7α-hydroxylase gene (CYP7A1), a key enzyme in bile acid biosynthesis, may influence the statin response. Methods and Results:To test this hypothesis, we examined a promoter polymorphism (A–290C) in CYP7A1 in 324 hypercholesterolemic patients treated with atorvastatin 10mg. The CYP7A1 polymorphism was significantly and independently associated with poor LDL cholesterol response. Mean reductions were -39% in wild type allele homozygotes, -37% in variant allele heterozygotes, and -34% in variant allele homozygotes, respectively (p<0.0001 for linear trend). The effects of this polymorphism were more striking in men than in women and were enhanced by the coexistence of common variants of the apolipoprotein E gene (APOE), ε2 or ε4. In subjects having wild type alleles at both loci, the mean reduction in LDL cholesterol was -40%, while in subjects having two CYP7A1 variant alleles and at least one variant APOE allele, the mean reduction in LDL cholesterol was -31% (p<0.0001). In addition, combination analysis of these two polymorphsims more accurately predicted the achievement of goal LDL cholesterol, than did both single polymorphism analysis. Conclusions:The CYP7A1 A–290C promoter variant was significantly and independently associated with poor response to atorvastatin. The effects of this polymorphism were additively enhanced, when common variants in another locus, APOE, coexist.
Plasma Sphingomyelin Levels in Coronary Artery Disease: A Relationship With Serum Triglyceride and Apolipoprotein B and Other Risk Factors
Axel Schlitt, Stefan Blankenberg, Daoguang Yan, Hans von Gyzicki, Christoph Bickel, Karl Lackner, Juergen Meyer, Hans J. Rupprecht, Xian-Cheng Jiang, State University of New York, Downstate Medical Center, Brooklyn, NY, Johannes Gutenberg University, Mainz, Germany Objective Sphingomyelin (SM) is one of the major phospholipids in the cell membrane and in lipoproteins. In human plasma, SM is mainly found in atherogenic lipoproteins, thus, high levels of SM may promote atherogenesis. Methods To further evaluate the role of SM in atherosclerosis, we measured plasma SM levels in 1,102 patients with coronary artery disease (CAD) and 444 healthy controls. Results We demonstrated that plasma SM in CAD patients was significantly higher than in controls (51.8 versus 44.9 mg/dl; p < 0.001). Logistic regression analysis showed that plasma SM was significantly associated with incidence of CAD (OR: 4.9, 95% CI 3.4 – 7.0, for subjects in the fourth quartile in comparison to subjects in the first quartile). Plasma SM levels showed a strongest and significant correlation with plasma apolipoprotein B (apoB)- (r=0.34, p < 0.001) and triglycerides -levels (r=0.31, p < 0.001) in all subjects, respectively. The association between SM and incidence of CAD remained independently significant after adjustment for most potential confounders including apoB and triglyceride, such that patients within the fourth quartile of SM revealed a 6.0 fold (95% CI 3.4 – 7.0) increase of risk. Conclusion These results reveal that the proatherogenic potency of human plasma SM levels, as shown in this study by significantly elevated levels of SM in patients, could be related to abnormal apoB -containing or triglyceride -rich lipoprotein metabolism.
ORAL CONTRIBUTIONS 841
Biology of Atherosclerosis: Inflammation and Plaque Instability Tuesday, March 09, 2004, 10:30 a.m.-Noon Morial Convention Center, Room 217 10:30 a.m. 841-1
Ceramide Triggers Weibel-Palade Body Exocytosis
Rinky Bhatia, Kenji Matsushita, Munekazu Yamakuchi, Craig Morrell, Charles J. Lowenstein, Johns Hopkins University School of Medicine, Baltimore, MD Background: The sphingolipid ceramide mediates a variety of stress responses, including vascular inflammation and thrombosis. Activated endothelial cells release Weibel-Palade bodies, granules containing vWF and P-selectin, which induce leukocyte rolling and platelet adhesion and aggregation. We hypothesized that ceramide induces vascular inflammation and thrombosis in part by triggering Weibel-Palade body exocytosis. Methods: We added ceramide to human aortic endothelial cells (HAEC) and assayed Weibel-Palade body exocytosis by measuring the concentration of vWF released into the media. Results: Exogenous ceramide induces vWF release from endothelial cells in a dosedependent manner. As little as 10 nM ceramide induces a 50% maximal release of vWF. Activators of endogenous ceramide production, neutral sphingomyelinase or tumor necrosis factor-a (TNF-a), also induce Weibel-Palade body exocytosis. We next studied the effects of nitric oxide (NO) on ceramide-induced Weibel-Palade body exocytosis, since NO can inhibit vascular inflammation. The NO donor S-nitroso-penicillamine (SNAP) decreases ceramide induced vWF release in a dose-dependent manner: the IC50% for SNAP inhibition of vWF release is < 1 uM. In contrast, the NOS inhibitor Lnitroarginine methyl ester (L-NAME) increases ceramide induced vWF release. Conclusions: In summary, our findings show that endogenous ceramide triggers WeibelPalade body exocytosis, and that endogenous NO inhibits ceramide induced exocytosis. These data suggest a novel mechanism by which ceramide induces vascular inflammation and thrombosis.
10:45 a.m. 841-2
Periadventitial Fat Inflammation Correlates With Plaque Inflammation in Patients With Coronary Plaque Ruptures: New Marker of Plaque Vulnerability?
Deborah Vela, Allen Burke, Alireza Zarrabi, Morteza Naghavi, Mohammad Madjid, Ward Casscells, III, Renu Virmani, James T. Willerson, Silvio Litovsky, Texas Heart Institute, Houston, TX, Armed Forces Institute of Pathology, Washington, DC Background. Previous work from our laboratory showed the presence of significant phagocytic activity in the aortic peri-adventitial fat of animal models of atherosclerosis (apoE-deficient mice and Watanabe rabbits). The macrophage activity is only mild in the aortic adventitial fat of wild-type mice and rabbits. In the present study we studied the adventitial fat of human coronary arteries in patients with plaque ruptures and contrasted them with those of patients with stable fibrocalcific plaques. We hypothesized that the macrophagic density would be much greater in the former.
Vascular Disease, Hypertension, and Prevention
Table. Best ‘Univariate’ Predictors of Insulin Sensitivity
1124-198
Vascular Disease, Hypertension, and Prevention
506A ABSTRACTS - Vascular Disease, Hypertension, and Prevention Material & Methods Ten histologic sections of human coronary plaque rupture were contrasted with ten cases of stable fibrocalcific plaques for the presence of peri-adventitial fat macrophages (Kp-1 positive cells). Quantitative morphometry was performed. Results. The ten plaque rupture cases had a markedly greater macrophage infiltration as evidenced by number/mm2 peri-adventitial fat. Moreover, the highest density in fibrocalcific cases (56/mm2) was lower than the lowest density in cases of plaque rupture (171/mm2). As expected, plaque rupture sections showed typical features of thin cap fibroatheroma with large lipid cores and high density of plaque macrophages that were accompanied by a significant macrophage infiltrate in the adventitia and peri-adventitial fat. S100 and Toluidine blue staining ruled out the possibility that these cells might represent dendritic cells or mast cells, respectively. Conclusion. This study shows the presence of active macrophages in the peri-adventitial fat of human coronary arteries. These cells were present in much greater quantity in the setting of plaque rupture as opposed to burnt-out fibrocalcific plaques. There appeared to be no significant difference in macrophage density between adventitia and peri-adventitial fat. Peri-adventitial fat inflammation might play a significant pathogenic role in atherosclerosis progression and complications.
11:00 a.m. Apolipoprotein A1 Mimetic Peptide Reduces Accelerated Atherosclerosis in a Vein Graft Bypass Model in ApoE (-/-) Mice
841-3
Xiaojun Li, Kuang-Yuh Chyu, Jose R. Faria Neto, Juliana Yano, Paul C. Dimayuga, Bojan Cercek, Prediman K. Shah, Cedars-Sinai Medical Center, Los Angeles, CA BACKGROUND: ApoA-1 mimetic peptide (DWFKAFYDKVAEKFKEAF or D4F) reduces diet-induced aortic atherosclerosis. Whether D4F is effective in reducing accelerated atherosclerosis in vein graft is unknown. We studied the effect of intraperitoneal (IP) administration of D4F on accelerated atherosclerosis in a murine vein graft bypass model. METHODS: Right carotid artery of hypercholesterolemic apoE (-/-) mice were grafted with a segment of IVC from donor mice at 16 weeks of age. Treatment group (n=10) received IP injection of 50 mcg D4F peptide daily for 4 weeks after surgery whereas control group (n=7) received saline injections. The grafts, heart and aorta were harvested and sectioned for morphometric and immunohistochemical analysis and plasma collected for cholesterol and D4F levels and lipoprotein fractionation analysis. RESULTS: D4F treatment significantly reduced plaque size in the vein graft but not in the aortic sinus or aorta. D4F also reduced lipid content in the vein graft but not in the aortic sinus plaques (Table). There were no difference in cholesterol levels or lipoprotein fractionation or plaque phenotypes in the vein graft or aortic sinus plaques between the groups. CONCLUSION: Four week treatment of Apo A-1 mimetic peptide reduces accelerated atherosclerosis in vein graft but has no effect on native spontaneous atherosclerosis in apoE (-/-) mice. Group
Cholesterol vein graft (mg/dl) plaque
lipid content aortic in vein graft sinus plaque size (mm2) (%) size
Lipid D-4F conc. content in (pmole/ml) aortic sinus (%)
(mm2) Control 1424±467
0.96±2.03
12.4±6.5
0.39±0.12
17.5±6.2
ND
D4F
0.55±0.37* 6.4±3.8*
0.50±0.09
18.0±6.6
3767±1183
1273±294
*: p<0.05, by t-test
11:15 a.m. 841-4
Bach1 Is a Key-Repressor of Heme Oxygenase-1 and Regulates Cell Proliferation
Shinji Omura, Jiying Sun, Hiroshi Suzuki, Kazuhiko Igarashi, Hiroshima University, Hiroshima-City, Japan Background: Heme oxygenase-1 (HO-1) protects cells from various insults including oxidative stress and its transcriptional induction by various stresses provides an important cellular adaptive defense mechanism. Recently we found that Bach1 is a physiological repressor of HO-1 (EMBO J., 2002). Though some investigators reported that HO-1 protects against vascular proliferation, the role for Bach1 in cell growth is poorly understood. The aim of this study is to elucidate the association between Bach1 and HO-1 expression and to examine the effect of bach1 ablation on cell proliferation. Methods: We have developed bach1 knockout (KO) mice and isolated aortic smooth muscle cells (SMC), macrophages, and embryonic fibroblasts (EF). Using RT-PCR method, western blotting and immunofluorescence staining, we analyzed the expression of HO-1 comparing them with that in wild type (WT) cells. Using retrovirus system we studied whether re-introduction of bach1 represses the expression HO-1 in KO cells or not. We also investigated effect of bach1 ablation on cell proliferation using BrdU labeling assay both atmospheric levels (20%), which is oxidative condition for EF, and physiological levels of oxygen (3%). We detected the senescent cells with senescence β-galactosidase staining. Results: Expression of HO-1 was increased in KO SMC, macrophages and EF as compared with WT cells (4.2-fold and p<0.01; 3.6-fold and p<0.05; 3.6-fold and p<0.01, respectively). While HO-1 expression was induced in WT cells when stimulated with Cd or hemin, no further induction was observed in the KO cells. Thus, HO-1 is fully activated in the absence of bach1 function. Expression of HO-1 was clearly inhibited in KO cells upon bach1 gene delivery. In 20% O2, proliferation of KO SMC and EF significantly decreased in both cell counting and in BrdU labeling assays. In 3% O2, KO EF prolifer-
JACC
March 3, 2004
ated as well as WT cells. When cultured in 20% O2, KO EF underwent senescence more frequently than WT cells. Conclusion: Inactivation of bach1 leads to increased expression of HO-1 and anti-proliferative effect. Bach1 is an important molecule for the response of oxidative stress. Bach1 may represent a novel molecular target in anti-atherosclerotic therapy.
11:30 a.m. 841-5
Attenuation of Human Atherosclerotic Plaque Inflammation by PPAR-γ Agonist and NF-κB Inhibitor
Tomasz Mazurek, LiFeng Zhang, Andrew Zalewski, Mark Kahn, Anthony Carabasi, Paul J. Dimusio, Rhoda Leichter, Ping Zhang, Anthony G. Johnson, Yi Shi, Thomas Jefferson University, Philadelphia, PA Background: Although inflammation plays an important role in formation, progression and instability of atherosclerotic plaque, its inflammatory properties have not been fully examined. Since the reduction of plaque inflammation may improve clinical outcomes in high-risk individuals, we examined inflammatory properties of human atherosclerotic plaque, and evaluate anti-inflammatory potential of PPAR-γ Agonist and NF-κB Inhibitor in an ex-vivo organ culture system utilizing advanced human atherosclerotic plaque for drug discovery. Methods: Human atherosclerotic plaques (CaP; n=20) were obtained during carotid endaterectomy. Unaffected vessels (radial arteries and saphenous veins: NV; n=8) were harvested during elective CABG surgery. Tissue expression of inflammatory mediators was analyzed by real-time RT-PCR (mRNA), and ELISA at baseline, after 2 and 24 hours incubation in the absence or presence of PPAR- γ agonist (rosiglitazone, 20 µM, n=14) or NK-κB inhibitor (MG-132, 100 µM, n=9). Results: At the baseline, CaP exhibited higher expression of IL-1β (7.8±2.1 vs. 1.9±0.4; p<0.01), TNF-α (0.9±0.1 vs. 0.3±0.1; p<0.01) and MCP-1 (0.9±0.1 vs. 0.3±0.1; p<0.01) on mRNA level, as compared to NV (respectively). After stimulation, rosiglitazone reduced TNF-α expression (4.4±0.9 vs. 2.3±0.4; p<0.05), whereas NFkB inhibitor attenuated TNFα (4.6±0.9 vs. 0.7±0.2; p<0.05), IL-6 (91.2±38 vs. 2.5±0.4; p<0.05), IL-8 (17.1±1.3 vs. 1.7±0.7; p<0.001), and MCP-1 (54.0±13.4 vs. 3.0±0.5; p<0.05) expression on protein and mRNA levels (not shown). Conclusions: 1. Atherosclerotic plaque releases higher levels of inflammatory mediators, as compared to normal vessel; 2. Inflammatory properties of atherosclerotic plaque are modifiable, that provides experimental platform for novel drug discovery in atherosclerosis; 3. Further evaluation of therapy targeting specific inflammatory pathways is necessary.
11:45 a.m. 841-6
Interleukin-18 and Interleukin-18 Binding Protein in Patients With Acute Coronary Syndromes
Craig R. Narins, David A. Lin, Zheng-Gen Jin, Bradford C. Berk, University of Rochester, Rochester, NY Background: Interleukin-18 (IL-18) is a pro-inflammatory cytokine produced by macrophages that induces interferon-γ production in T-cells, and acts with IL-12 to upregulate Thelper cell activity. In animal models IL-18 is a potent promoter of both atherogenesis and plaque instability, and among patients with coronary disease serum IL-18 is a predictor of subsequent cardiovascular death. IL-18 binding protein (IL-18BP) is a recently identified circulating high-affinity antagonist that binds to and neutralizes IL-18. We sought determine (1) The strength of the relationship between serum IL-18 concentration and clinical coronary events, and (2) the nature of the relationship between IL-18 and IL-18BP among patients with coronary artery disease. Methods: Serum concentrations of IL-18, IL-18BP and other inflammatory mediators (IL5, IL-8, IL-12, IL-13, anti-HSP60, GM-CSF, TNF, MCP-1, IFN-γ) were measured immediately prior to coronary angioplasty in 79 patients. Patients were grouped as acute coronary syndrome positive (ACS+) or negative (ACS-) based on presence or absence of elevated troponin I levels. Results: No significant differences existed between ACS+ and ACS- patients with respect to clinical variables (including age, gender, prior MI, smoking, cholesterol, hypertension, diabetes). The IL-18 concentration was significantly greater among ACS+ vs. ACSpatients (452 ± 384 vs. 301 ± 172 pg/ml, p=0.017). There was, however, no significant difference between the groups with respect to IL-18BP levels, as reflected by an elevated ratio of IL-18 / IL-18BP among the ACS+ compared to ACS- patients (150 ± 145 vs. 89 ± 67, p=0.011). Of all mediators examined, only IL-18 was significantly associated with the presence of recent ACS. Conclusions: IL-18 emerged as the most potent correlate of recent plaque instability among the mediators and clinical variables examined. The elevated serum IL-18 concentrations present in the ACS+ patients were not “opposed” by a concomitant rise in IL18BP levels. These findings support a clinical association between IL-18 and plaque instability, and support further study of IL-18BP as a potential means to modulate the pro-inflammatory effects of IL-18.
March 3, 2004
ABSTRACTS - Vascular Disease, Hypertension, and Prevention 505A
R2
increased to 0.44 (P<0.05). Finally, a model which included WHR, Head fat, and Upper Body fat yielded a maximal R2 of 0.47 (P<0.05), an improvement over either set of measurements alone. Conclusion: Simple measures when used alone are as effective as more sophisticated methods like DEXA for predicting SI in a healthy population. However, when these measures are combined there is a small increased predictive power.
Parameter
Estimate
t Ratio
Pr > t
R2
Weight (kg)
-0.020
-11.47
<0.0001
0.37
BMI
-0.062
-12.77
<0.0001
0.40
Head % Fat
-0.040
-12.52
<0.0001
0.41
Upper Body Fat (kg)
-0.044
-12.50
<0.0001
0.41
Head Fat (kg)
-0.807
-12.78
<0.0001
0.42
Waist (cm)
-0.028
-13.38
<0.0001
0.42
* Age and gender were forced into every model.
1124-196
Impact of Elevated Age and Sex-Adjusted Body Mass Index in School Age Children on Insulin Resistance and Lipoprotein Subfractions
David K. Murdock, Karen Olson, Kirk Jenkins, Mary Roth, Sharon Fox, Patrick Hughes, Bryan Hendricks, Cardiovascular Research and Education Foundation, Inc, Wausau, WI, Community Health Care/Wausau Health Foundation, Wausau, WI Background: The Wausau SCHOOL Project is a community-based effort to assess the frequency of cardiovascular risk factors in students in the Wausau School District. Objective: Define the incidence of elevated BMI and determine its relationship to standard lipid profiles, homeostatic model assessment of insulin resistance (HOMA-IR), LDL particle size and number. Methods: Age and sex adjusted BMI (zBMI), fasting plasma insulin, glucose levels and nuclear magnetic resonance lipid profiles (LipoScience®) was measured in 225 randomly selected students (110 in 2nd grade and 125 in 11th grade). Overweight was defined as zBMI > 85th centile based on 2000 CDC norms. Results: Over 31% of the students were classified as overweight. (30.6% in 2nd grade and 32.5% in 11th grade). HOMA-IR values were significantly higher for 11th graders than for 2nd graders (2.18±2.65 vs. 1.22± 1.07, p<0.01). HOMA-IR values were also significantly higher for females than for males (2.04±2.66 vs. 1.33±0.94, p<0.05). Those with zBMI above the 85th centile had significantly higher levels of total cholesterol (182.0±34.0 vs. 170.2±28.5, p<0.01), LDL (123.4±28.5 vs. 112.7±25.7, p<0.01), triglycerides (118.6±80.7 vs. 86.4±35.6, p<0.001), LDL particle number (1299.8±379.5 vs. 1135±248.0, p<0.001), and HOMA-IR (2.61±3.47 vs. 1.32±0.09, p< 0.001), while HDL levels were lower (43.7±10.0 vs. 47.5 ±9.7, p<0.01). There were no significant differences in LDL particle size. Conclusions: A significant percentage of children and adolescents are overweight. IR increases significantly with age and zBMI and is associated with an atherogenic lipid profile characterized by an increase in the number of small LDL particles and TG levels and lower HDL levels, similar to that seen in adults.
1124-197
Additive Gene-Gene Interaction Between CYP7A1 and Apolipoprotein E as Genetic Determinants of LowDensity Lipoprotein Cholesterol-Lowering Response to Atorvastatin
Kouji Kajinami, Noboru Takekoshi, Margaret E. Brousseau, Jose M. Ordovas, Ernst J. Schaefer, Kanazawa Medical University, Uchinada, Japan, Tufts University, Boston, MA Background: The mechanisms responsible for interindividual variation in response to statin therapy remain uncertain. Bile acid biosynthesis is one of the determinants of intracellular cholesterol and, in turn, cholesterol synthesis rate in hepatocytes. This raises the hypothesis that variation in the cholesterol 7α-hydroxylase gene (CYP7A1), a key enzyme in bile acid biosynthesis, may influence the statin response. Methods and Results:To test this hypothesis, we examined a promoter polymorphism (A–290C) in CYP7A1 in 324 hypercholesterolemic patients treated with atorvastatin 10mg. The CYP7A1 polymorphism was significantly and independently associated with poor LDL cholesterol response. Mean reductions were -39% in wild type allele homozygotes, -37% in variant allele heterozygotes, and -34% in variant allele homozygotes, respectively (p<0.0001 for linear trend). The effects of this polymorphism were more striking in men than in women and were enhanced by the coexistence of common variants of the apolipoprotein E gene (APOE), ε2 or ε4. In subjects having wild type alleles at both loci, the mean reduction in LDL cholesterol was -40%, while in subjects having two CYP7A1 variant alleles and at least one variant APOE allele, the mean reduction in LDL cholesterol was -31% (p<0.0001). In addition, combination analysis of these two polymorphsims more accurately predicted the achievement of goal LDL cholesterol, than did both single polymorphism analysis. Conclusions:The CYP7A1 A–290C promoter variant was significantly and independently associated with poor response to atorvastatin. The effects of this polymorphism were additively enhanced, when common variants in another locus, APOE, coexist.
Plasma Sphingomyelin Levels in Coronary Artery Disease: A Relationship With Serum Triglyceride and Apolipoprotein B and Other Risk Factors
Axel Schlitt, Stefan Blankenberg, Daoguang Yan, Hans von Gyzicki, Christoph Bickel, Karl Lackner, Juergen Meyer, Hans J. Rupprecht, Xian-Cheng Jiang, State University of New York, Downstate Medical Center, Brooklyn, NY, Johannes Gutenberg University, Mainz, Germany Objective Sphingomyelin (SM) is one of the major phospholipids in the cell membrane and in lipoproteins. In human plasma, SM is mainly found in atherogenic lipoproteins, thus, high levels of SM may promote atherogenesis. Methods To further evaluate the role of SM in atherosclerosis, we measured plasma SM levels in 1,102 patients with coronary artery disease (CAD) and 444 healthy controls. Results We demonstrated that plasma SM in CAD patients was significantly higher than in controls (51.8 versus 44.9 mg/dl; p < 0.001). Logistic regression analysis showed that plasma SM was significantly associated with incidence of CAD (OR: 4.9, 95% CI 3.4 – 7.0, for subjects in the fourth quartile in comparison to subjects in the first quartile). Plasma SM levels showed a strongest and significant correlation with plasma apolipoprotein B (apoB)- (r=0.34, p < 0.001) and triglycerides -levels (r=0.31, p < 0.001) in all subjects, respectively. The association between SM and incidence of CAD remained independently significant after adjustment for most potential confounders including apoB and triglyceride, such that patients within the fourth quartile of SM revealed a 6.0 fold (95% CI 3.4 – 7.0) increase of risk. Conclusion These results reveal that the proatherogenic potency of human plasma SM levels, as shown in this study by significantly elevated levels of SM in patients, could be related to abnormal apoB -containing or triglyceride -rich lipoprotein metabolism.
ORAL CONTRIBUTIONS 841
Biology of Atherosclerosis: Inflammation and Plaque Instability Tuesday, March 09, 2004, 10:30 a.m.-Noon Morial Convention Center, Room 217 10:30 a.m. 841-1
Ceramide Triggers Weibel-Palade Body Exocytosis
Rinky Bhatia, Kenji Matsushita, Munekazu Yamakuchi, Craig Morrell, Charles J. Lowenstein, Johns Hopkins University School of Medicine, Baltimore, MD Background: The sphingolipid ceramide mediates a variety of stress responses, including vascular inflammation and thrombosis. Activated endothelial cells release Weibel-Palade bodies, granules containing vWF and P-selectin, which induce leukocyte rolling and platelet adhesion and aggregation. We hypothesized that ceramide induces vascular inflammation and thrombosis in part by triggering Weibel-Palade body exocytosis. Methods: We added ceramide to human aortic endothelial cells (HAEC) and assayed Weibel-Palade body exocytosis by measuring the concentration of vWF released into the media. Results: Exogenous ceramide induces vWF release from endothelial cells in a dosedependent manner. As little as 10 nM ceramide induces a 50% maximal release of vWF. Activators of endogenous ceramide production, neutral sphingomyelinase or tumor necrosis factor-a (TNF-a), also induce Weibel-Palade body exocytosis. We next studied the effects of nitric oxide (NO) on ceramide-induced Weibel-Palade body exocytosis, since NO can inhibit vascular inflammation. The NO donor S-nitroso-penicillamine (SNAP) decreases ceramide induced vWF release in a dose-dependent manner: the IC50% for SNAP inhibition of vWF release is < 1 uM. In contrast, the NOS inhibitor Lnitroarginine methyl ester (L-NAME) increases ceramide induced vWF release. Conclusions: In summary, our findings show that endogenous ceramide triggers WeibelPalade body exocytosis, and that endogenous NO inhibits ceramide induced exocytosis. These data suggest a novel mechanism by which ceramide induces vascular inflammation and thrombosis.
10:45 a.m. 841-2
Periadventitial Fat Inflammation Correlates With Plaque Inflammation in Patients With Coronary Plaque Ruptures: New Marker of Plaque Vulnerability?
Deborah Vela, Allen Burke, Alireza Zarrabi, Morteza Naghavi, Mohammad Madjid, Ward Casscells, III, Renu Virmani, James T. Willerson, Silvio Litovsky, Texas Heart Institute, Houston, TX, Armed Forces Institute of Pathology, Washington, DC Background. Previous work from our laboratory showed the presence of significant phagocytic activity in the aortic peri-adventitial fat of animal models of atherosclerosis (apoE-deficient mice and Watanabe rabbits). The macrophage activity is only mild in the aortic adventitial fat of wild-type mice and rabbits. In the present study we studied the adventitial fat of human coronary arteries in patients with plaque ruptures and contrasted them with those of patients with stable fibrocalcific plaques. We hypothesized that the macrophagic density would be much greater in the former.
Vascular Disease, Hypertension, and Prevention
Table. Best ‘Univariate’ Predictors of Insulin Sensitivity
1124-198
Vascular Disease, Hypertension, and Prevention
506A ABSTRACTS - Vascular Disease, Hypertension, and Prevention Material & Methods Ten histologic sections of human coronary plaque rupture were contrasted with ten cases of stable fibrocalcific plaques for the presence of peri-adventitial fat macrophages (Kp-1 positive cells). Quantitative morphometry was performed. Results. The ten plaque rupture cases had a markedly greater macrophage infiltration as evidenced by number/mm2 peri-adventitial fat. Moreover, the highest density in fibrocalcific cases (56/mm2) was lower than the lowest density in cases of plaque rupture (171/mm2). As expected, plaque rupture sections showed typical features of thin cap fibroatheroma with large lipid cores and high density of plaque macrophages that were accompanied by a significant macrophage infiltrate in the adventitia and peri-adventitial fat. S100 and Toluidine blue staining ruled out the possibility that these cells might represent dendritic cells or mast cells, respectively. Conclusion. This study shows the presence of active macrophages in the peri-adventitial fat of human coronary arteries. These cells were present in much greater quantity in the setting of plaque rupture as opposed to burnt-out fibrocalcific plaques. There appeared to be no significant difference in macrophage density between adventitia and peri-adventitial fat. Peri-adventitial fat inflammation might play a significant pathogenic role in atherosclerosis progression and complications.
11:00 a.m. Apolipoprotein A1 Mimetic Peptide Reduces Accelerated Atherosclerosis in a Vein Graft Bypass Model in ApoE (-/-) Mice
841-3
Xiaojun Li, Kuang-Yuh Chyu, Jose R. Faria Neto, Juliana Yano, Paul C. Dimayuga, Bojan Cercek, Prediman K. Shah, Cedars-Sinai Medical Center, Los Angeles, CA BACKGROUND: ApoA-1 mimetic peptide (DWFKAFYDKVAEKFKEAF or D4F) reduces diet-induced aortic atherosclerosis. Whether D4F is effective in reducing accelerated atherosclerosis in vein graft is unknown. We studied the effect of intraperitoneal (IP) administration of D4F on accelerated atherosclerosis in a murine vein graft bypass model. METHODS: Right carotid artery of hypercholesterolemic apoE (-/-) mice were grafted with a segment of IVC from donor mice at 16 weeks of age. Treatment group (n=10) received IP injection of 50 mcg D4F peptide daily for 4 weeks after surgery whereas control group (n=7) received saline injections. The grafts, heart and aorta were harvested and sectioned for morphometric and immunohistochemical analysis and plasma collected for cholesterol and D4F levels and lipoprotein fractionation analysis. RESULTS: D4F treatment significantly reduced plaque size in the vein graft but not in the aortic sinus or aorta. D4F also reduced lipid content in the vein graft but not in the aortic sinus plaques (Table). There were no difference in cholesterol levels or lipoprotein fractionation or plaque phenotypes in the vein graft or aortic sinus plaques between the groups. CONCLUSION: Four week treatment of Apo A-1 mimetic peptide reduces accelerated atherosclerosis in vein graft but has no effect on native spontaneous atherosclerosis in apoE (-/-) mice. Group
Cholesterol vein graft (mg/dl) plaque
lipid content aortic in vein graft sinus plaque size (mm2) (%) size
Lipid D-4F conc. content in (pmole/ml) aortic sinus (%)
(mm2) Control 1424±467
0.96±2.03
12.4±6.5
0.39±0.12
17.5±6.2
ND
D4F
0.55±0.37* 6.4±3.8*
0.50±0.09
18.0±6.6
3767±1183
1273±294
*: p<0.05, by t-test
11:15 a.m. 841-4
Bach1 Is a Key-Repressor of Heme Oxygenase-1 and Regulates Cell Proliferation
Shinji Omura, Jiying Sun, Hiroshi Suzuki, Kazuhiko Igarashi, Hiroshima University, Hiroshima-City, Japan Background: Heme oxygenase-1 (HO-1) protects cells from various insults including oxidative stress and its transcriptional induction by various stresses provides an important cellular adaptive defense mechanism. Recently we found that Bach1 is a physiological repressor of HO-1 (EMBO J., 2002). Though some investigators reported that HO-1 protects against vascular proliferation, the role for Bach1 in cell growth is poorly understood. The aim of this study is to elucidate the association between Bach1 and HO-1 expression and to examine the effect of bach1 ablation on cell proliferation. Methods: We have developed bach1 knockout (KO) mice and isolated aortic smooth muscle cells (SMC), macrophages, and embryonic fibroblasts (EF). Using RT-PCR method, western blotting and immunofluorescence staining, we analyzed the expression of HO-1 comparing them with that in wild type (WT) cells. Using retrovirus system we studied whether re-introduction of bach1 represses the expression HO-1 in KO cells or not. We also investigated effect of bach1 ablation on cell proliferation using BrdU labeling assay both atmospheric levels (20%), which is oxidative condition for EF, and physiological levels of oxygen (3%). We detected the senescent cells with senescence β-galactosidase staining. Results: Expression of HO-1 was increased in KO SMC, macrophages and EF as compared with WT cells (4.2-fold and p<0.01; 3.6-fold and p<0.05; 3.6-fold and p<0.01, respectively). While HO-1 expression was induced in WT cells when stimulated with Cd or hemin, no further induction was observed in the KO cells. Thus, HO-1 is fully activated in the absence of bach1 function. Expression of HO-1 was clearly inhibited in KO cells upon bach1 gene delivery. In 20% O2, proliferation of KO SMC and EF significantly decreased in both cell counting and in BrdU labeling assays. In 3% O2, KO EF prolifer-
JACC
March 3, 2004
ated as well as WT cells. When cultured in 20% O2, KO EF underwent senescence more frequently than WT cells. Conclusion: Inactivation of bach1 leads to increased expression of HO-1 and anti-proliferative effect. Bach1 is an important molecule for the response of oxidative stress. Bach1 may represent a novel molecular target in anti-atherosclerotic therapy.
11:30 a.m. 841-5
Attenuation of Human Atherosclerotic Plaque Inflammation by PPAR-γ Agonist and NF-κB Inhibitor
Tomasz Mazurek, LiFeng Zhang, Andrew Zalewski, Mark Kahn, Anthony Carabasi, Paul J. Dimusio, Rhoda Leichter, Ping Zhang, Anthony G. Johnson, Yi Shi, Thomas Jefferson University, Philadelphia, PA Background: Although inflammation plays an important role in formation, progression and instability of atherosclerotic plaque, its inflammatory properties have not been fully examined. Since the reduction of plaque inflammation may improve clinical outcomes in high-risk individuals, we examined inflammatory properties of human atherosclerotic plaque, and evaluate anti-inflammatory potential of PPAR-γ Agonist and NF-κB Inhibitor in an ex-vivo organ culture system utilizing advanced human atherosclerotic plaque for drug discovery. Methods: Human atherosclerotic plaques (CaP; n=20) were obtained during carotid endaterectomy. Unaffected vessels (radial arteries and saphenous veins: NV; n=8) were harvested during elective CABG surgery. Tissue expression of inflammatory mediators was analyzed by real-time RT-PCR (mRNA), and ELISA at baseline, after 2 and 24 hours incubation in the absence or presence of PPAR- γ agonist (rosiglitazone, 20 µM, n=14) or NK-κB inhibitor (MG-132, 100 µM, n=9). Results: At the baseline, CaP exhibited higher expression of IL-1β (7.8±2.1 vs. 1.9±0.4; p<0.01), TNF-α (0.9±0.1 vs. 0.3±0.1; p<0.01) and MCP-1 (0.9±0.1 vs. 0.3±0.1; p<0.01) on mRNA level, as compared to NV (respectively). After stimulation, rosiglitazone reduced TNF-α expression (4.4±0.9 vs. 2.3±0.4; p<0.05), whereas NFkB inhibitor attenuated TNFα (4.6±0.9 vs. 0.7±0.2; p<0.05), IL-6 (91.2±38 vs. 2.5±0.4; p<0.05), IL-8 (17.1±1.3 vs. 1.7±0.7; p<0.001), and MCP-1 (54.0±13.4 vs. 3.0±0.5; p<0.05) expression on protein and mRNA levels (not shown). Conclusions: 1. Atherosclerotic plaque releases higher levels of inflammatory mediators, as compared to normal vessel; 2. Inflammatory properties of atherosclerotic plaque are modifiable, that provides experimental platform for novel drug discovery in atherosclerosis; 3. Further evaluation of therapy targeting specific inflammatory pathways is necessary.
11:45 a.m. 841-6
Interleukin-18 and Interleukin-18 Binding Protein in Patients With Acute Coronary Syndromes
Craig R. Narins, David A. Lin, Zheng-Gen Jin, Bradford C. Berk, University of Rochester, Rochester, NY Background: Interleukin-18 (IL-18) is a pro-inflammatory cytokine produced by macrophages that induces interferon-γ production in T-cells, and acts with IL-12 to upregulate Thelper cell activity. In animal models IL-18 is a potent promoter of both atherogenesis and plaque instability, and among patients with coronary disease serum IL-18 is a predictor of subsequent cardiovascular death. IL-18 binding protein (IL-18BP) is a recently identified circulating high-affinity antagonist that binds to and neutralizes IL-18. We sought determine (1) The strength of the relationship between serum IL-18 concentration and clinical coronary events, and (2) the nature of the relationship between IL-18 and IL-18BP among patients with coronary artery disease. Methods: Serum concentrations of IL-18, IL-18BP and other inflammatory mediators (IL5, IL-8, IL-12, IL-13, anti-HSP60, GM-CSF, TNF, MCP-1, IFN-γ) were measured immediately prior to coronary angioplasty in 79 patients. Patients were grouped as acute coronary syndrome positive (ACS+) or negative (ACS-) based on presence or absence of elevated troponin I levels. Results: No significant differences existed between ACS+ and ACS- patients with respect to clinical variables (including age, gender, prior MI, smoking, cholesterol, hypertension, diabetes). The IL-18 concentration was significantly greater among ACS+ vs. ACSpatients (452 ± 384 vs. 301 ± 172 pg/ml, p=0.017). There was, however, no significant difference between the groups with respect to IL-18BP levels, as reflected by an elevated ratio of IL-18 / IL-18BP among the ACS+ compared to ACS- patients (150 ± 145 vs. 89 ± 67, p=0.011). Of all mediators examined, only IL-18 was significantly associated with the presence of recent ACS. Conclusions: IL-18 emerged as the most potent correlate of recent plaque instability among the mediators and clinical variables examined. The elevated serum IL-18 concentrations present in the ACS+ patients were not “opposed” by a concomitant rise in IL18BP levels. These findings support a clinical association between IL-18 and plaque instability, and support further study of IL-18BP as a potential means to modulate the pro-inflammatory effects of IL-18.